Rapid detection of ethambutol-resistant Mycobacterium tuberculosis strains by PCR-RFLP targeting embB codons 306 and 497 and iniA codon 501 mutations
Introduction
Ethambutol [dextro-2,2′-(ethylenediimino) di-1-butanol] (EMB) is a bactericidal, first-line drug for the treatment of tuberculosis (TB). EMB along with isoniazid, rifampin and pyrazinamide is used as an alternative to streptomycin, in the directly observed therapy short-course anti-TB regimen recommended by the World Health Organization and the Center for Disease Control and Prevention (CDC). Recent global data on drug resistance showed that resistance of Mycobacterium tuberculosis to streptomycin and EMB in newly diagnosed cases varied from 1 to 32% and 0 to 11%, respectively [1]. The primary resistance of M. tuberculosis strains to streptomycin and EMB in the United States was shown to be 6 and 2%, respectively [1]. The prevalence of primary and acquired resistance to EMB is generally much lower than that for streptomycin and is predominant among isolates from patients with multidrug-resistant-TB (MDR-TB). The early start of an effective therapy with at least two drugs to which the strain is susceptible is a key requirement for a positive treatment outcome in MDR-TB [2]. EMB is thus a valuable and alternative drug for streptomycin. Since resistance to EMB is generally associated with resistance to other antitubercular drugs, an early detection of embB resistance will not only abolish the risk of adverse reactions associated with EMB, particularly optic neuritis, it will also indicate the need to modify the therapy regimen.
Although the mechanism of action and the molecular genetic basis of resistance to EMB are complex and not fully defined, the drug interacts mainly with the membrane associated arabinosyltransferases as an arabinose analogue [3], [4]. The M. tuberculosis emb operon contains three contiguous genes namely embC, embA and embB encoding three homologous arabinosyltransferases [4]. Earlier studies involving EMB-resistant M. tuberculosis strains identified resistance-conferring amino acid substitutions in the embB gene in nearly 50 to 70% of the isolates [3], [4], [5]. The nucleotide substitutions most commonly occurred at codon position 306ATG-Met of embB gene. Five different mutations were found in this codon that altered its first or third base (ATG to GTG, CTG, ATA, ATC or ATT) resulting in the replacement of methionine by three different amino acids (Met to Val, Leu or Ile) [5]. Subsequent studies carried out on epidemiologically unrelated EMB-resistant M. tuberculosis strains not only showed the involvement of many more codons of the three arabinosyltransferases but also several other genes in EMB resistance [6]. However, this study also showed that the most frequent mutations occurred at embB codons 306, 406 and 497 and iniA codon 501 [6].
Many EMB-resistant M. tuberculosis strains can be rapidly identified by molecular methods that interrogate embB codons 306, 406 and 497 and iniA codon 501 [4], [5], [6]. However, elaborate and expensive methods such as DNA sequencing have been mostly used [4], [5], [6]. In this report, we describe simple and easily interpretable PCR-restriction fragment length polymorphism (RFLP) methods to detect, directly in BACTEC cultures, the most frequently found embB codons 306 and 497 and iniA codon 501 mutations in EMB-resistant M. tuberculosis strains.
Section snippets
Reference strains, clinical samples and DNA isolation
The M. tuberculosis H37Rv was used as the susceptible strain and well-characterized M. tuberculosis strains with specific substitutions at embB codons 306 and 497 and iniA codon 501, established by DNA sequencing of the DNA region around the respective codon positions, were used as reference strains. Twenty-five M. tuberculosis strains, isolated from TB patients, were obtained from the Chest Diseases Hospital, Kuwait. Isolation and identification of the clinical isolates was performed as
Results
The PCR performed on the DNA from the susceptible strain M. tuberculosis H37Rv (Fig. 1, Panel A, Lane Rv) and the mutant strains carrying ATG to GTG (Fig. 1, Panel A, Lane Mu1) or ATG to ATT (Fig. 1, Panel A, Lane Mu2) or ATG to CTG (Fig. 1, Panel A, Lane Mu3) mutation at embB codon 306 with primers EMB306A and EMB306B resulted in specific amplification of a 167 bp DNA fragment, as expected. The Nla III digested fragments of the amplified DNA from M. tuberculosis H37Rv resolved into four
Discussion
EMB was introduced as an effective anti-TB drug in the 1960s and is now used as an alternative drug for streptomycin. The worldwide prevalence of resistance to EMB is much lower than for streptomycin and is also predominant among patients with MDR-TB from some geographical locations/ethnic groupings [1]. Several studies have shown that majority of EMB-resistant M. tuberculosis strains contain mutations at embB codons 306, 406 and 497 and iniA codon 501 [4], [5], [6]. In particular, mutations at
Acknowledgements
We thank Z. Khan for technical assistance. This work was supported by Research Administration grant MI 06/02 and the College of Graduate Studies, Kuwait University.
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